Malaria causes almost half a million deaths each year. Existing interventions will almost certainly not be enough to tackle this enormous public health problem on their own. An effective vaccine is urgently needed. The leading malaria vaccine, RTS,S, confers suboptimal protective efficacy, and in addition targets only Plasmodium falciparum and not the other major species of human malaria, P. vivax. This thesis investigates the potential of combining pre-erythrocytic malaria vaccines as a means of enhancing protective efficacy. A novel mathematical model was developed which expresses probability of protection as a function of vaccine-induced humoural and cellular responses. The model predicts that combining partially effective vaccines should result in more than additive improvements in protective efficacy. This was supported by an experiment combining Rv21, a P. vivax circumsporozoite virus-like particle, with viral vectored P. vivax TRAP, the two leading pre-erythrocytic malaria vaccine antigens; this combination raised protective efficacy from 50% and 0%, respectively, to 100% sterile protection. It was also found that antigenic interference, a reduction in anti-CSP titres when Rv21 and PvTRAP are combined, occurred only in the presence of Matrix M adjuvant, and not when using alum, AddaVax or no adjuvant. With a view to creating a single-component multi-antigen vaccine, which would be more cost-effective than a multi-component vaccine, experiments were carried out to establish the virus-like particle Qβ as a platform capable of eliciting protective immunity via the display of short peptides derived from the CSP repeat region of both P. vivax and P. falciparum. For the first time, a tetramer peptide derived from the CSP repeat region of P. vivax VK210, AGDR, was shown capable of eliciting protective immunity alone. Finally, five novel linear B-cell epitopes were discovered, one from P. falciparum CSP, three from P. vivax TRAP and one from TRSP, each capable of conferring partial protection on mice. These epitopes were identified using novel screening methods, using sera from whole-protein vaccinated mice or by exploiting conservation within invasion protein sequences. Two of the protective epitopes, (NANP)6 and (ADGN long) were combined and found to enhance protective efficacy as predicted by the mathematical model. Thus this thesis lays the groundwork for the development of a single-component multi-epitope malaria vaccine with enhanced protective efficacy.

Plasmodium falciparum, the most pestilential of the malaria parasite species, is responsible for ~450,000 direct deaths annually. Clinical disease is a consequence of the blood stage of the parasite’s lifecycle involving a plethora of host-parasite interactions. Key to these interactions are the P. falciparum reticulocyte binding-like homologue (PfRh) proteins responsible for binding erythrocyte receptors and gaining entry to host cells. For example, PfRh4 binds to human complement receptor-1 (CR1) on erythrocytes for sialic-acid-independent invasion. Another protein important for invasion is the PfRh5-interacting protein (PfRipr), an essential member of the PfRh5-associated invasion complex (PAIN-complex) along with CyRPA, the cysteine-rich protective antigen. Loss of function of PfRipr in P. falciparum parasites prevents erythrocyte entry and ablates Ca2+-influx into the erythrocyte; essential events during invasion. This study aimed to biochemically and structurally investigate truncated recombinant versions of PfRh4 and PfRipr. Homology modelling suggested that PfRh4 is rich in alpha-helical secondary structure. The sequence of PfRipr suggested the presence of ten epidermal growth factor-like (EGF) modules, two towards the N-terminus and eight in the C-terminal domain. In this project, monoclonal antibodies made against recombinant PfRh4 were shown, via indirect immunofluorescent assays, to localize to the apical tip of merozoites. Monoclonal antibody 5H12, raised against PfRh4, reduces parasite invasion of erythrocytes by ~75% in growth-inhibition assays with neuraminidase pre-treated erythrocytes. Attempts to produce a stable truncated recombinant PfRh4 protein for structural studies were unsuccessful. An ELISA-based assay using ten alanine-scan mutants suggested the CR1-binding site lies outside of amino acids 283 – 341 of PfRh4. PfRipr truncations, defined by the boundaries of EGF-like repeats predicted based on sequence homology, were produced recombinantly in Escherichia coli and Pichia pastoris. These proteins had a circular dichroism signature suggestive of β-strand-containing proteins with disordered regions. EGF-containing PfRipr truncations did not bind recombinant PfRh5 according to ELISA and size-exclusion chromatography assays. EGFs 1-2, 5-7 and 7-10 of PfRipr did not bind CyRPA via size-exclusion chromatography or NMR. Crystallisation trials performed on EGF modules failed to yield crystals suitable for data collection. A 15N isotopically-labelled sample of EGF5-7 gave good quality HSQC NMR spectra. A suite of three-dimensional NMR spectra collected on a 13C,15N-EGF5-7 sample, at three different temperatures, allowed for >86% of backbone assignments. T1/T2 relaxation analysis and heteronuclear NOE data were suggestive of an elongated, rigid protein undergoing intermolecular self-association. Further evidence for EGF5-7 being an elongated protein was provided via SAXS analysis. Chemical shifts facilitated prediction of secondary structure in EGF 5-7 consistent with an EGF-like fold. Melting studies performed on EGF5-7 showed no evidence of denaturation over the temperature range 20 °C - 95 °C indicating a thermally-stable protein. The addition of Ca2+ to the 15N-EGF5-7 sample caused chemical shift perturbations consistent with high-affinity binding. The discovery of inhibitory monoclonal antibodies recognising a conformational epitope on EGF7 provided evidence of the functional importance of this region within PfRipr. The work described in this thesis provides methods for the industrially-scalable production and biophysical investigations of P. pastoris or E. coli-produced disulfide-rich P. falciparum antigens of interest to vaccinologists.

Thesis (Ph. D.)--University of Missouri-Columbia, 1999. / Typescript. Vita. Includes bibliographical references (leaves 52-53). Also available on the Internet.

Diss. (sammanfattning) Stockholm : Univ., 2006. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Missouri-Columbia, 1999. / Typescript. Vita. Includes bibliographical references (leaves 52-53). Also available on the Internet.

Apical membrane antigen 1 (AMA1) is a leading candidate vaccine antigen against blood stage malaria and several clinical trials using mostly protein-in-adjuvant vaccines have shown limited success. This thesis describes the development of recombinant adenoviral (AdHu5) and poxviral (MVA) vectors encoding AMA1 from Plasmodium chabaudi murine parasites. In this murine malaria model, AdHu5 and MVA encoding AMA1 when used in a heterologous prime boost regime showed excellent immunogenicity, both humoral and cellular. The vaccination regime was protective against blood stage challenge and both antibodies and CD4+ T cells found to be important for vaccine induced blood stage protection. In parallel to this novel P. falciparum vaccines encoding AMA1 were also developed and administered in a similar prime boost regime to mice and rabbits. The vaccination regime induced cellular immune response and high titre antibodies against AMA1 and these antibodies showed growth inhibitory activity against the homologous parasite strain. In an effort to overcome the issue of antigenic polymorphism and to circumvent pre-existing immunity to human adenovirus, biallelic simian and human adenoviral vectors and MVA encoding AMA1 vaccines were also developed and administered to mice and macaques. These vectors also induced high titre antibodies and the serum from macaques was found to have growth inhibitory activity. These vaccine candidates are now being taken forward to Phase I/II clinical trials in Oxford. This work also described the attempt to improve MVA as a antibody inducing vector to allow better antibody mediated immunity to blood stage malaria.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Miu Fei Fei. / On t.p. "42" are subscripts following the word "MSP-1" in the title. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 117-125). / Abstracts in English and Chinese. / ACKNOWLEGEMENTS --- p.i / ABSTRACT --- p.ii / TABLE OF CONTENTS --- p.v / LIST OF FIGURE --- p.vii / LIST OF ABBREVIATIONS --- p.ix / CHAPTERS: / Chapter 1. --- BACKGROUND OF MALARIA / Chapter 1.1 --- Epidemilogy --- p.2 / Chapter 1.2 --- Mode of Infection --- p.4 / Chapter 1.3 --- Conventional Control & Vaccination --- p.9 / Chapter 1.4 --- Vaccine Candidate PfMSV-142 --- p.16 / Chapter 1.5 --- Cloning and Expression of pfMSP-142 --- p.26 / Chapter 1.6 --- Aims of Study --- p.32 / Chapter 2. --- Materials and Methods / Chapter 2.1 --- Materials --- p.32 / Chapter 2.2 --- Methods --- p.38 / Chapter 3. --- Construction of recombinants N-PfMSP-142 and C- PfMSP-142 / Chapter 3.1 --- Construction of C-PfMSP-l42 --- p.51 / Chapter 3.2 --- Construction ofN-PfMSP-l42 --- p.56 / Chapter 4. --- Purification with IMAC / Chapter 4.1 --- Immobilized Metal Affinity Chromatography (IMAC) --- p.58 / Chapter 4.2 --- Purification ofN-PfMSP-l42 --- p.61 / Chapter 4.3 --- Purification profile of N-PfMSP-142 --- p.68 / Chapter 4.4 --- Purification of C-PfMSP-l42 --- p.70 / Chapter 4.5 --- Purification profile of C-PfMSP-142 --- p.73 / Chapter 4.6 --- Expression pattern of recombinants PfMSP-142 --- p.76 / Chapter 5. --- Purification combined with other chromatography method / Chapter 5.1 --- Affinity chromatography --- p.78 / Chapter 5.2 --- Gel filtration chromatography --- p.80 / Chapter 5.3 --- Ion exchange chromatography --- p.83 / Chapter 5.4 --- Conclusion --- p.93 / Chapter 6. --- Characteristic of IMAC products --- p.94 / Chapter 7. --- Characteristic of N-hisPfMSP-l42 & C-hisPfMSP-l --- p.42 / Chapter 7.1 --- Immunogenitcity of N-PfMSP-l42 and C-PfMSP-142 --- p.100 / Chapter 7.2 --- Competitive ELISA --- p.105 / Chapter 8. --- Discussion --- p.107 / REFERENCE --- p.117

In the second part of the project, the immunology of PfMSP-133 was studied. During the invasion of merozoites, PfMSP--142 is processed into two fragments with molecular weight of 33kDa and 19kDa. The 19kDa fragment (PfMSP-119) originating from the carboxyl--terminal of PfMSP--142 is relatively more immuno-dominant in different malarial species such as P. falciparum, P. vivax and P. yoelii. In the past, only limited researches about PfMSP-1 33 were performed. Apart from its difficulty in expression, PfMSP-1 33 was also believed to be incapable of inducing protection. / Nevertheless, following the breakthrough of expressing recombinant PfMSP-1 33 in our laboratory, we have demonstrated in this study that recombinant MSP-133 can elicit antibodies with a titer up to a million. Also, we observed that MSP-133 can help MSP-119 to induce protective immunity and such effect is independent from the covalent linkage between these two proteins. Most importantly, our results show that recombinant PfMSP-133 can elicit the production of antibodies that can potentiate the inhibitory effect of anti-MSP-142 serum at high serum dilution. Results of this study give new insights in malarial vaccine development in terms of optimizing the use of adjuvant and immunization regimens. / The 42kDa carboxyl terminal fragment of Plasmodium falciparum Merozoite Surface Protein-1 (PfMSP--142) is one of the most promising candidate antigens in the development of malarial vaccine. In vivo experiments in the 1990's showed that Aotus monkeys immunized with PfMSP--142 were protected from malarial challenge. Later on, other experiments also demonstrated the possibility of using recombinant PfMSP-142 as candidate antigen for malarial vaccine. Previously, recombinant PfMSP-142 (Bvp42) was expressed with the baculovirus expression system and characterized in our laboratory. / The aim of the first part of this project is to improve the production of Bvp42. Experimental results have shown that the expression level of Bvp42 was increased under a BMN compatible baculovirus expression vector---pVL1393. Besides, a codon optimized MSP-142 nucleotide is constructed for the construction of a baculovirus carrying codon optimized MSP-142 gene and aimed for higher expression level. Unfortunately, no Bvp42 expression is observed in the transfection samples and the reason of this observation is unclear. Meanwhile, the purification of Bvp42 was also improved. Pretreatment of the hemolymph with Q--sepharose before affinity chromatography could enhance the purity of the final product. / Yuen, Sai-hang Don. / "July 2007." / Adviser: Walter K. K. Ho. / Source: Dissertation Abstracts International, Volume: 69-01, Section: B, page: 0220. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 183-195). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on July 19, 2010). Includes bibliographical references.